蚕豆叶片下表皮ABA结合蛋白提取及分离条件的选择

以蚕豆（ＶｉｃｉａｆａｂａＬ．）叶片下表皮为材料,比较ＴｒｉｔｏｎＸ１００、冷丙酮和（ＮＨ４）２ＳＯ４对ＡＢＡ结合蛋白（简称ＡＢＡＢＰ）的提取效果。结果表明：０．５％（Ｗ／Ｖ）ＴｒｉｔｏｎＸ１００去垢剂提取的ＡＢＡＢＰ与ＡＢＡ特异结合活性较高（０．４８７ｎｍｏｌ／ｇｐｒｏｔｅｉｎ）,维持结合活性的时间较长（４℃下反应４０ｈ保持最大结合的６０％）;而冷丙酮法提取的ＡＢＡＢＰ特异结合活性只有０．３２５ｎｍｏｌ／ｇｐｒｏｔｅｉｎ,且容易失活,１０ｈ仅保持最大结合的３０％左右。实验比较了各种盐离子对ＡＢＡＢＰ的影响,高盐（＞３００ｍｍｏｌ／ＬＮａＣｌ）不利于ＡＢＡＢＰ的结合反应,低浓度ＫＣｌ对ＡＢＡＢＰ活性略有促进。ＡＢＡＢＰ的结合活性需要介质中有一定量的Ｃａ２＋和Ｍｇ２＋,用ＥＤＴＡ螯合介质中Ｍｇ２＋、Ｃａ２＋后,ＡＢＡＢＰ活性大大降低,分别为最大结合的７５％和６０％。ＡＢＡＢＰ与ＡＢＡ反应的最适ｐＨ在６．５,这些条件为亲和层析纯化ＡＢＡＢＰ提供了依据。     

Isolation and Purification of ABA Binding Protein from Abaxial Epiderm of Vicia faba Leaf

Abscisic acid (ABA) was efficiently cross-linked to Sepharose 4B (6 ~8 mmol ABA/L gel) by an ann of 10-atom carbon chain. Solubilized ABA-BP (ABA binding protein) was allowed to bind to the gel, while unrelated proteins were removed by washing with a gradient of NaC1 buffer. The ABA-BP was eluted with 1 mmol/L ABA. Since ABA at high concemration can interfere with both the binding activity assay and protein analysis, the fractions eluted with ABA were passed through a Sephadex G-25 column to remove the ABA. Fractions containing the binding activity were pooled, concentrated with uhm-fihration. The maximum binding capacity (BMAX) of the purified ABA-BP was 58.33 nmol/g protein, and the Kd was 21 nmol/L, with an approximately 112 folds increase of purity. SDS-PAGE identification of the purified ABA-BP revealed a major protein band with a molecular weight of about 44.2 kD, and a purity of approximately 90 %.     

Partial Purification of an Ethylene-binding Component from Plant Tissue

An ethylene binding component(s) has been partially purified from mung bean sprouts. Tissue was homogenized in 0.3 molar sucrose and 0.2 molar potassium phosphate buffer (pH 7.0). The homogenate was centrifuged, and resuspended fractions were assayed by incorporating them onto cellulose fibers (0.7 grams per milliliter). These were exposed to [¹⁴C]ethylene (3.7 × 10⁻² microliters per liter of 120 millicurie per millimole) in the presence or absence of 1000 microliters per liter unlabeled ethylene. The cellulose was transferred to separate containers and the [¹⁴C]ethylene was absorbed in mercury perchlorate and counted. Distribution of ethylene binding to various fractions was: 0 to 3,000g, 3%; 3,000 to 12,000g; 4%; 12,000 to 100,000g, 69%; cellular debris, 24%; 100,000g supernatant, 0%. Adjustment of the pH to 4.0 precipitates the ethylene-binding component. Neutralization, addition of Triton X-100, and readjustment of the pH to 4.0 “solubilized” most of the binding component. Further purification was obtained by chromatography on CM-Sephadex in 10 millimolar potassium acetate buffer, (pH 5.0) containing 1% Triton X-100. Elution was with 200 millimolar potassium phosphate (pH 6.0) containing 1% Triton X-100. Upon treatment of the Triton “solubilized” component with cold acetone, over 90% of the binding capacity was lost. Extraction of the acetone-precipitated residue with 2% Triton X-100 restored some of the binding capacity which was found in the soluble fraction. The pH optimum for binding is 6.0. Passing the Triton X-100 extract of the acetone powder through Sepharose 6B provides considerable purification. The binding component moved ahead of most of the protein.     

葡萄果实中脱落酸结合蛋白的存在及其性质

葡萄果实微粒体上存在高亲和力的脱落酸（ＡＢＡ）结合位点,这些位点与ＡＢＡ的结合具有饱和性,高亲和力及低容量,胰蛋白酶或ＤＴＴ处理可以使该位点的特异结合活性下降约９０％,表明此结合位点是一种蛋白质,故称为ＡＢＡ结合蛋白,它含有维系蛋白质特定构象的二硫键,该蛋白与ＡＢＡ反应的最适ｐＨ为６．０,说明与配基结合部位可能存在带有正电荷的氨基酸残基,结合活性在２５℃高于０℃,结合反应达到动态平衡需要３０ｍｉｎ,３０ｍｉｎ以后结合活性随时间延长而下降。该蛋白与ＡＢＡ结合反应的平衡解离常数为１７．５ｎｍｏｌ／Ｌ,最大结合容量（Ｂｍａｘ）为９８．４ｆｍｏｌ／ｍｇｐｒｏｔｅｉｎ。     

Subcellular Localization and Kinetic Characterization of ABA Binding Proteins in Maize Root

By means of differential centrifugation, cytosol fraction and microsome were prepared from maize roots which have been grown in dark for 4 d. Highly purified plasma membranes were isolated from the microsome in two-phase aqueous system which is composed of 6.9 % (W/W) Dextran T500 and PEG 3350. The tonoplast was collected from the interface between 1% and 8% (W/W) Dextran T70 after gradient centrifugation. Electron microscopic observation and marker enzyme activities analysis proved that these fractions contained very few other membranes. Microvolume radioactivity ligand binding assay indicated that the specific binding sites of ABA in maize root microsome were mainly distributed on tonoplast and plasma membrane fractions. Their specific binding activity was 2485.4 and 1257.3 fmol/mg protein, respectively, the specific binding activity of cytosol fraction being the lowest (one order of magnitude lower). The dissociation constant (KD) of ABA-BP in plasma membrane was 1.57 nmol/L.     

Properties of acid ceramidase from human spleen

We have characterised ceramidase activity in extracts of human spleen from control subjects and from patients with Gaucher disease. In Triton X-100 extracts of control spleens, a broad pH optimum of pH 3.5-5.0 was found; no ceramidase activity was detectable at neutral or alkaline pH. About 45-60% of acid ceramidase could be extracted from spleen without detergents, but for complete extraction, Triton X-100 was required. For the radiolabelled substrate oleoylsphingosine, a Km of 0.22 +/- 0.09 mM and a Vmax of 57 +/- 11 nmol/h per mg protein was calculated in spleen from a control subject. Flat-bed isoelectric focussing in the presence of Triton X-100 revealed a pI of 6.0-7.0 for acid ceramidase; similar values were found for sphingomyelinase and glucerebrosidase. HPLC-gel filtration indicated that in the presence of Triton X-100, acid ceramidase has an Mr of about 100 kDa. In the absence of detergents, the enzyme forms high-molecular-weight aggregates. Similar aggregation behaviour was observed for sphingomyelinase, while the elution of beta-hexosaminidase was not affected by detergents. The elution profile of glucocerebrosidase was only slightly altered by Triton X-100. There was no difference in the properties of acid ceramidase present in spleen from control subjects and from patients with type I Gaucher disease.     

Factors effecting the solubilisation of stearoyl-coA desaturase of hen liver microsomes.

1. The lipid requirement for maximum desaturase activity was investigated using acetone/water mixtures. It was shown that for maximum stearoyl-CoA desaturase activity of hen liver microsomes neither the total neutral lipid fraction nor 44% of the phospholipid fraction were required. 2. The effect of sodium deoxycholate, Triton X-100, Nonidet P-40 and Bio-solv on the enzyme activity indicated that the neutral detergents had a milder effect than the ionic detergent but both classes could cause considerable irreversible loss of activity. 3. The treatment of the microsomes with 2.5% (v/v) water in acetone greatly improved the effective solubilising power of Triton X-100. The yield of desaturase in the 100 000 X g supernatant obtained by treating the microsomal fraction in this way was strongly dependent upon protein concentration. Maximum solubilisation was achieved with25 mg protein per ml 1% (w/v) Triton X-100 in 0.1 M potassium phosphate buffer pH 7.4. 4. A comparison of the properties of the solubilised and membrane-bound enzyme was made by an investigation of: (i) the temperature and pH optimum, (ii) activation energy and (iii) the effect of inhibitors on the enzyme activity.     

Galactosylation of endogenous proteins from human platelets

Human platelets have been shown to contain the enzyme glycoprotein:galactosyltransferase that catalyzes the transfer of galactose to an endogenous protein acceptor present in the platelet. Galactosylation of added ovalbumin also occurs. The activity was extracted with 30 mM Tris buffer (pH 7.5). The endogenous activity was enriched 1.4-fold (compared with the crude homogenate) in the fraction, 105,000 g pellet, and the exogenous enzyme was retained in the respective supernatant. The two galactosyltransferase activities showed proportionality to time, protein, and substrate concentration, and were identical in pH dependence and Mn+2 requirement. The effect of Triton X-100 (range 0-1.5%) in the assay system appeared to be different for both activities: with the optimum concentration of detergent (0.15%) the endogenous activity increased by 50% whereas the exogenous activity was augmented 5-fold. From a number of sugar nucleotides tested as glycosyl donor into the endogenous proteins, the optimum substrate was UDP-Glc (100%), followed by UDP-Gal (80%), GDP-Man (24%), UDP-Glc-NAc (21%), UDP-Xyl (19%), and ADP-Glc (5%). An appropriate exogenous acceptor for UDP-Glc as donor was not found. The different solubilization of galactosyl- and glucosyltransferase activities by Triton X-100 suggests that they are distinct enzymes. In addition, the exogenous galactosyltransferase activity achieved after the treatment was much higher (940%) than the endogenous (26%). It is suggested that these differences on both galactosyltransferases could reflect changes in the accessibility of the exogenous substrate to the enzyme.     

Comparison of platelet-derived growth factor prepared from release products of fresh platelets and from outdated platelet concentrates

Treatment of synaptic membranes from rat brainstem and spinal cord with the nonionic detergent Triton X-100 at 1-10 microliters/mg protein caused a marked increase in glycine receptor (3H)strychnine binding expressed per mg of residual membrane protein. The effect was maximal (220 +/- 6% of control) at 5 microliters Triton/mg protein, while higher concentrations caused progressive loss of strychnine binding ability of membranes (27 +/- 6% at 25 microliters Triton/mg protein). The increase in strychnine binding caused by low Triton X-100 reflected an increase in membrane Bmax, the kD being unaffected by the treatment. The affinity of glycine analogues for receptor sites was not appreciably affected by the detergent either. The findings suggest an enrichment of the synaptic membrane preparation in glycine receptors, caused by the solubilization by Triton of membrane constituents not related to the receptor sites.     

ISOLATION AND CHARACTERIZATION OF SOLUBLE ACIDIC LIPOPROTEINS FROM RAT BRAIN SYNAPTIC VESICLES

—Highly purified fractions of synaptic vesicles were prepared from rat cerebrum or cerebral cortex by density gradient centrifugation. Treatment of synaptic vesicle fractions by autoincubation, freeze-thawing and sonication in an isotonic alkaline-salt medium or in 0·1-0·3% (v/v) Triton X-100 released increasing quantities of synaptic vesicle protein and phospholipid into solution. When the soluble synaptic vesicle proteins were extracted with 0·1% (v/v) Triton X-100, the insoluble residue consisted mostly of 5–8 nm-thick membranes resembling the limiting membranes of intact synaptic vesicles. This finding, together with other considerations, suggested that the soluble proteins and accompanying phospholipids originated from the interior of the synaptic vesicles. A 0·3% (v/v) Triton X-100 extract of synaptic vesicle was fractionated by ultracentrifugal flotation and dialysis into three lipoprotein fractions: a low density lipoprotein (d < 1·21 g/ml), a high density lipoprotein (d = 1·21–1·35 g/ml) and a very high density lipoprotein (d > 1·35 g/ml). The phospholipid contents of the low, high and very high density lipoprotein fractions were 0·74, 0·38 and 0·20 mg/mg of protein, respectively. All three apolipoproteins had a high ratio of acidic to basic, and of polar to nonpolar, amino acids, and were rich in glycine, alanine and serine. Polyacrylamide gel electrophoresis of the alkaline-salt and Triton X-100 extracts of synaptic vesicles at pH 8·8 resolved a single anionic component which stained for protein, lipid (Sudan black B; iodine) and anionic groups (acridine orange). Polyacrylamide gel electrophoresis of synaptic vesicle extracts at pH 2·7 in 5 m urea and 0·25% (v/v) Triton X-100 resolved about 20 protein components. However, the protein profiles of electropherograms of the Triton X-100 and alkaline-salt extracts differed in certain respects, suggesting that these media to some extent solubilized different proteins. However, most of the protein bands in electropherograms of the Triton X-100 and alkaline-salt extracts also stained for lipid and anionic groups. In addition, two lipoprotein components in the alkaline-salt extract and four in the Triton X-100 extract contained carbohydrate. Isoelectric focusing of synaptic vesicle extracts resolved 6–8 protein fractions. The major fraction in Triton X-100 and alkaline-salt extracts had an apparent isoelectric point of approximately 4·2 and contained 0·24 mg of phospholipid per mg of protein. Soluble synaptic vesicle proteins released by incubating, freeze-thawing and sonicating in the alkaline-salt medium, and protein fractions of the latter obtained by electrofocusing had an absorption maximum of 260–265 nm which was enhanced in a cold 0·5 n perchloric acid extract, an observation suggesting the presence of a bound nucleotide. These findings demonstrate that rat brain synaptic vesicles contain a heterogenous array of soluble acidic lipoproteins which vary in buoyant density, lipid content, amino acid and carbohydrate composition and electrophoretic mobility in polyacrylamide gels. These acidic lipoproteins apparently comprise the bulk of the macromolecular contents of synaptic vesicles and probably serve as ‘carrier’ proteins for the binding and sequestration of the neurotransmitters.